The dramatic increase of genome sequences across multiple species is exponentially increasing the number of predicted proteins with unknown function. The traditional one at a time approach of characterizing factors will always be essential, but it helps to be able to have a global view of the functional connections between and among these proteins in order to aid in the generation of specific hypotheses. One important way to study protein function is to be able to ascertain which other proteins are associated with it. In this proposal, we aim to systematically generate a protein-protein interaction (PPI) data in the model organism, S. pombe. Next to budding yeast, the fission yeast S. pombe has the most easily manipulatable genome of any eukaryotic model organism, and yet it is evolutionarily distinct from S. cerevisiae. We aim to generate the fission yeast PPI map using a two-pronged platform: yeast two-hybrid methodology and co-affinity purification followed by mass spectrometry (AP/MS) approach. This work brings together two individuals (Yu and Krogan) who worked together to generate the most comprehensive co-complex PPI map to date in any organism (S. cerevisiae) using the AP/MS strategy (Krogan et al., Nature, 2006). Furthermore, more recently, Yu generated the most comprehensive two-hybrid dataset to date, also in budding yeast (Yu et al., Science, 2008). Therefore, we argue that, between the two groups, we have the experimental and bioinformatic expertise to carry out a comprehensive analysis of the protein interactome in fission yeast. From a purely evolutionary standpoint, valuable information would be obtained by comparing the interactome networks from both organisms since these two yeasts diverged around 330-420 million years ago. Additionally, analysis of interaction networks in S. pombe will provide novel information that cannot be obtained from studies performed with S. cerevisiae. For example, in its large complex centromere structure, the restriction of spindle construction to mitotic entry, redundant and inefficient origins of replication, gene regulation by histone methylation and chromodomain heterochromatin proteins, gene and transposon regulation by the RNAi pathway, telomere binding proteins, and the presence of introns in most genes, S. pombe is more similar to metazoans than is S. cerevisiae. Therefore, this project will not only have a huge impact on the fission yeast field but also will fuel evolutionary studies and will allow for interrogation of biological systems that are present in higher organisms but not in budding yeast.